Liquid chromatography is a process utilized in both preparative and analytical chemistry. Essentially, the process comprises a stationary phase interacting with a mobile phase. Typically, the stationary phase is a surface-active powder such as silica, alumina, or an inert size-separating material like a gel-permeation chromatography packing, or the like. This powder is contained in a chromatographic column. A mobile phase, generally consisting of a carrier fluid and a sample of a chemical to be identified, analyzed, or purified, is passed through the column. A typical utility of the process is to identify various chemical components in an unknown sample. This identification is made by (1) using an immobile phase which differentially retards the progress of different components of the sample through the column so that the components are separated and leave the column at different times and (2) continuously analyzing the effluent of the column over a period of time. The separation is achieved when one component of the sample has more affinity for the stationary phase than does another component. Also, the separation may be achieved by an exclusion process based on the difference in sizes between molecules, e.g. by gel-permeation chromatography processes. The invention to be described below is related to achieving better and more dependable identity of the sample components by improving the efficiency of the process in such a way as to provide better resolution of the sample.
In order to achieve separation of sample components which are very close to each other in chemical and physical properties, highly sophisticated procedures have been developed in the many processing techniques associated with liquid chromatography. Special pumps and valves have been developed for presenting samples to the inlet of the chromatographic columns with as much integrity as is possible to avoid building into the process an initial and inherent dispersal of the sample which dispersal would tend to reduce the resolution capabilities of the chromatographic packing within the column. Moreover, much work has been done to provide flow-distributing devices at the inlet of the column to assure the even placement of the sample across the columns cross-sectional area. Also, a great deal of technical effort has provided improved chromatographic packings and highly-sophisticated analytical apparatus for measuring various properties of the liquid effluent leaving the column.
Despite such work as has been described above, it has remained a problem to achieve an uniform packing of the chromatographic material into a column. Many techniques have been suggested including vibration (See U.S. Pat. No. 3,300,849): All of these techniques require careful control if segregation of particles by size is to be avoided and uniformly packed columns are to be obtained. Even after the column is filled, problems exist in maintaining the filling in proper condition during transportation and operation of the packed columns. (See U.S. Pat. No. 3,349,920 to Waters.)
In general, the most commonly used practice of filling a high-performance column has been a costly method including slurrying the packing and passing the slurry into the column; thereby, in effect, using the column itself as a form for placing a "filter cake" of chromatographic packing therein. Each this costly, time-consuming method of column manufacture is not without problems caused by shifting of the packing during shipment when it can be subjected to various vibration and other transient, non-predictable physical abuse. This tends to cause voids in the column and such voids can wholly destroy a column's operating characteristics for many separations. Such defects in stainless steel columns are not usually detectable until a standard sample is measured as a control. Indeed, suppliers of quality chromatographic columns, until this day, have pre-tested individually each column before shipment to the customer to assure that the packing is properly placed in the column. Of course, this "certifying procedure" provides no protection against the hazards encountered during shipment or during use by the customer.
A number of solutions have been suggested for holding the packing "in-place". Some of these, like the aforementioned vibration technique and slurrying technique, emphasize a maximum effort to put a conventional packing into the column in such a way as to have it assume a stable position. Other techniques such as those described in U.S. Pat. No. 3,808,125 to Good use rather complex or expensive procedures for fastening the packing to the column wall.
None of these attempts by the prior art have been dependably successful in achieving any of an excellent performance, a column-to-column consistency in separating characteristics, or a desired degree of stability of performance over a period of time for a single column, at a cost which can make the apparatus available to the broadest spectrum of chromatographers.
Although the foregoing description of problems relating to chromatographic columns has been largely devoted to liquid chromatographic columns, it is emphasized that many of the problems described above also relate to gas chromatography, i.e. chromatography wherein the sample and mobile phase are in gaseous, rather than liquid, form. Indeed, in many respects, the problems relate to all packed-bed apparatus comprising a porous mass of particles intended to be intimately and uniformly contacted by a fluid. Such apparatus includes catalytic beds for the treating of gas and liquid, packed beds used in ion exchange processes, in electrophoresis applications, and the like. It is intended that the invention described below be viewed as an improvement in packed-column-preparation for all such processes; albeit, the invention will be seen to have particular advantage in the field of liquid chromatography.
In discussing packed-column processes, it is helpful to recognize four kinds of space, all of which can be referred to as "void volume". These include (1) void volume inside a porous particle; (2) theoretical void volume between particles, i.e. the type of unavoidable volume which would result from a perfectly packed bed of spheres of the same size; (3) void volume which is attributable to imperfect packing of particles, usually present to some extent in any actual system utilizing a particulate-packing system; and (4) void volume which represents relatively large voids resulting from the consolidation of those voids described in (3). Voids (4) substantially reduce resolution of a sample being subjected to chromatographic analysis.
The invention to be disclosed below is believed to be most useful in avoiding the occurrence of such void volume as described in (4). The present invention also tends to reduce void volume as described in (3); moreover, it makes such void volume more nearly uniform, and closer to a theoretical ideal. Void volume, as generally used herein relates to a composite of void volumes (3) and (4).
Some workers have suggested compression of the packing of a chromatographic column by force directed longitudinally, i.e. parallel to the direction of liquid flow. However, such a procedure is relatively ineffective probably because the packing tends to bridge the column and interfere with propagation of the compression force downwardly throughout the length of the column. An example of such work is described in the Journal of Chromatographic Science of October, 1974, in an article entitled "Description and Performance of an 8 cm i.d. Column For Preparative Scale High Pressure Liquid-Solid Chromatography" by Godbille and Devaux.
The above discussion of the Background of the Invention is made, necessarily, in view of the Applicants' invention to be described below. It should be understood that the collection, interpretation and discussion of this background is not intended to disclose the background from the point of view of one being ordinarily skilled in the art and having no preliminary knowledge of Applicant's invention.